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1.
Environ Toxicol Pharmacol ; 59: 13-16, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29482112

RESUMO

The possible role of voltage-sensitive calcium channels (VSCC) activation on the HgCl2-induced dopamine release was investigated using selective VSCC blockers and the dopamine levels were measured by HPLC from samples obtained by in vivo brain microdialysis. Infusion of HgCl2 in nicardipine (10 or 100 µM) or flunaricine (10 µM) pretreated animals had no significant effect on dopamine release induced by HgCl2. Pretreatment with 100 µM flunaricine, 20 µM ω-conotoxin MVIIC, or ω-conotoxin GVIA significantly decreased the HgCl2-induced dopamine release over 61%, 88%, and 99%, respectively. HgCl2-induced dopamine release could be produced, at least in part, by activation of VSCC at dopaminergic terminals, especially N- and P/Q-type.


Assuntos
Canais de Cálcio/metabolismo , Corpo Estriado/efeitos dos fármacos , Dopamina/metabolismo , Mercúrio/toxicidade , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Corpo Estriado/metabolismo , Feminino , Flunarizina/farmacologia , Nicardipino/farmacologia , Ratos Sprague-Dawley , ômega-Conotoxina GVIA/farmacologia , ômega-Conotoxinas/farmacologia
2.
Neuron ; 96(6): 1317-1326.e4, 2017 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-29198756

RESUMO

Action potential induces membrane depolarization and triggers intracellular free Ca2+ concentration (Ca2+)-dependent secretion (CDS) via Ca2+ influx through voltage-gated Ca2+ channels. We report a new type of somatic exocytosis triggered by the action potential per se-Ca2+-independent but voltage-dependent secretion (CiVDS)-in dorsal root ganglion neurons. Here we uncovered the molecular mechanism of CiVDS, comprising a voltage sensor, fusion machinery, and their linker. Specifically, the voltage-gated N-type Ca2+ channel (CaV2.2) is the voltage sensor triggering CiVDS, the SNARE complex functions as the vesicle fusion machinery, the "synprint" of CaV2.2 serves as a linker between the voltage sensor and the fusion machinery, and ATP is a cargo of CiVDS vesicles. Thus, CiVDS releases ATP from the soma while CDS releases glutamate from presynaptic terminals, establishing the CaV2.2-SNARE "voltage-gating fusion pore" as a novel pathway co-existing with the canonical "Ca2+-gating fusion pore" pathway for neurotransmitter release following action potentials in primary sensory neurons.


Assuntos
Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Cálcio/metabolismo , Ativação do Canal Iônico/genética , Células Receptoras Sensoriais/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Cafeína/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/ultraestrutura , Exocitose/efeitos dos fármacos , Exocitose/genética , Gânglios Espinais/citologia , Gânglios Espinais/ultraestrutura , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Masculino , Fusão de Membrana/efeitos dos fármacos , Fusão de Membrana/genética , Modelos Moleculares , Inibidores de Fosfodiesterase/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Terminações Pré-Sinápticas/fisiologia , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Proteínas SNARE/genética , Proteínas SNARE/metabolismo , Células Receptoras Sensoriais/ultraestrutura , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/genética , Transdução Genética , ômega-Conotoxina GVIA/farmacologia
3.
Zhongguo Zhong Yao Za Zhi ; 42(6): 1118-1124, 2017 Mar.
Artigo em Chinês | MEDLINE | ID: mdl-29027426

RESUMO

Apocynum venetum belongs to apocynaceae and is a perennial medicinal plant, its stem is an important textile raw materials. The projection of potential geographic distribution of A. venetum has an important significance for the protection and sustainable utilization of the plant. This study was conducted to determine the potential geographic distribution of A. venetum and to project how climate change would affect its geographic distribution. The projection geographic distribution of A. venetum under current bioclimatic conditions in northern China was simulated using MaxEnt software based on species presence data at 44 locations and 19 bioclimatic parameters. The future distributions of A. venetum were also projected in 2050 and 2070 under the climate change scenarios of RCP2.6 and RCP8.5 described in 5th Assessment Report of the Intergovernmental Panel on Climate Change (IPCC). The result showed that min air temperature of the coldest month, annual mean air temperature, precipitation of the coldest quarter and mean air temperature of the wettest quarter dominated the geographic distribution of A. venetum. Under current climate, the suitable habitats of A. venetum is 11.94% in China, the suitable habitats are mainly located in the middle of Xinjiang, in the northern part of Gansu, in the southern part of Neimeng, in the northern part of Ningxia, in the middle and northern part of Shaanxi, in the southern part of Shanxi, in the middle and northern part of Henan, in the middle and southern part of Hebei, Shandong, Tianjin, in the southern part of Liaoning and part of Beijing. From 2050 to 2070, the model outputs indicated that the suitable habitats of A. venetum would decrease under the climate change scenarios of RCP2.6 and RCP8.5.


Assuntos
Apocynum/crescimento & desenvolvimento , Mudança Climática , Ecossistema , China , Previsões , ômega-Conotoxina GVIA
4.
Endocrinology ; 158(10): 3426-3434, 2017 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-28938466

RESUMO

Incretin hormones play an important role in the regulation of food intake and glucose homeostasis. Glucagonlike peptide-1 (GLP-1)-secreting cells have been demonstrated to be electrically excitable and to fire action potentials (APs) with increased frequency in response to nutrient exposure. However, nutrients can also be metabolized or activate G-protein-coupled receptors, thus potentially stimulating GLP-1 secretion independent of their effects on the plasma membrane potential. Here we used channelrhodopsins to manipulate the membrane potential of GLUTag cells, a well-established model of GLP-1-secreting enteroendocrine L cells. Using channelrhodopsins with fast or slow on/off kinetics (CheTA and SSFO, respectively), we found that trains of light pulses could trigger APs and calcium elevation in GLUTag cells stably expressing either CheTA or SSFO. Tetrodotoxin reduced light-triggered AP frequency but did not impair calcium responses, whereas further addition of the calcium-channel blockers nifedipine and ω-conotoxin GVIA abolished both APs and calcium transients. Light pulse trains did not trigger GLP-1 secretion from CheTA-expressing cells under basal conditions but were an effective stimulus when cyclic adenosine monophosphate (cAMP) concentrations were elevated by forskolin plus 3-isobutyl 1-methylxanthine. In SSFO-expressing cells, light-stimulated GLP-1 release was observed at resting and elevated cAMP concentrations and was blocked by nifedipine plus ω-conotoxin GVIA but not tetrodotoxin. We conclude that cAMP elevation or cumulative membrane depolarization triggered by SSFO enhances the efficiency of light-triggered action potential firing, voltage-gated calcium entry, and GLP-1 secretion.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Células Enteroendócrinas/efeitos dos fármacos , Peptídeo 1 Semelhante ao Glucagon/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , 1-Metil-3-Isobutilxantina/farmacologia , Animais , Cálcio/metabolismo , Colforsina/farmacologia , Células Enteroendócrinas/metabolismo , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Camundongos , Nifedipino/farmacologia , Optogenética , Técnicas de Patch-Clamp , Inibidores de Fosfodiesterase/farmacologia , Rodopsina , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Vasodilatadores/farmacologia , ômega-Conotoxina GVIA/farmacologia
5.
Neuron ; 95(4): 914-927.e4, 2017 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-28781167

RESUMO

Compartmentalized signaling in dendritic subdomains is critical for the function of many central neurons. In the retina, individual dendritic sectors of a starburst amacrine cell (SAC) are preferentially activated by different directions of linear motion, indicating limited signal propagation between the sectors. However, the mechanism that regulates this propagation is poorly understood. Here, we find that metabotropic glutamate receptor 2 (mGluR2) signaling, which acts on voltage-gated calcium channels in SACs, selectively restricts cross-sector signal propagation in SACs, but does not affect local dendritic computation within individual sectors. mGluR2 signaling ensures sufficient electrotonic isolation of dendritic sectors to prevent their depolarization during non-preferred motion, yet enables controlled multicompartmental signal integration that enhances responses to preferred motion. Furthermore, mGluR2-mediated dendritic compartmentalization in SACs is important for the functional output of direction-selective ganglion cells (DSGCs). Therefore, our results directly link modulation of dendritic compartmentalization to circuit-level encoding of motion direction in the retina.


Assuntos
Células Amácrinas/citologia , Células Amácrinas/fisiologia , Dendritos/fisiologia , Percepção de Movimento/fisiologia , Retina/citologia , Transdução de Sinais/fisiologia , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/fisiologia , Aminoácidos/farmacologia , Animais , Animais Recém-Nascidos , Cloreto de Cádmio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Dendritos/efeitos dos fármacos , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estimulação Luminosa , Receptores de AMPA/genética , Receptores de AMPA/metabolismo , Retina/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Sinapses/efeitos dos fármacos , Sinapses/fisiologia , Xantenos/farmacologia , ômega-Conotoxina GVIA/farmacologia
6.
J Neurosci ; 37(7): 1757-1771, 2017 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-28087765

RESUMO

Synapsins are epilepsy susceptibility genes that encode phosphoproteins reversibly associated with synaptic vesicles. Synapsin II (SynII) gene deletion produces a deficit in inhibitory synaptic transmission, and this defect is thought to cause epileptic activity. We systematically investigated how SynII affects synchronous and asynchronous release components of inhibitory transmission in the CA1 region of the mouse hippocampus. We found that the asynchronous GABAergic release component is diminished in SynII-deleted (SynII(-)) slices. To investigate this defect at different interneuron subtypes, we selectively blocked either N-type or P/Q-type Ca2+ channels. SynII deletion suppressed the asynchronous release component at synapses dependent on N-type Ca2+ channels but not at synapses dependent on P/Q-type Ca2+ channels. We then performed paired double-patch recordings from inhibitory basket interneurons connected to pyramidal neurons and used cluster analysis to classify interneurons according to their spiking and synaptic parameters. We identified two cell subtypes, presumably parvalbumin (PV) and cholecystokinin (CCK) expressing basket interneurons. To validate our interneuron classification, we took advantage of transgenic animals with fluorescently labeled PV interneurons and confirmed that their spiking and synaptic parameters matched the parameters of presumed PV cells identified by the cluster analysis. The analysis of the release time course at the two interneuron subtypes demonstrated that the asynchronous release component was selectively reduced at SynII(-) CCK interneurons. In contrast, the transmission was desynchronized at SynII(-) PV interneurons. Together, our results demonstrate that SynII regulates the time course of GABAergic release, and that this SynII function is dependent on the interneuron subtype.SIGNIFICANCE STATEMENT Deletion of the neuronal protein synapsin II (SynII) leads to the development of epilepsy, probably due to impairments in inhibitory synaptic transmission. We systematically investigated SynII function at different subtypes of inhibitory neurons in the hippocampus. We discovered that SynII affects the time course of GABA release, and that this effect is interneuron subtype specific. Within one of the subtypes, SynII deficiency synchronizes the release and suppresses the asynchronous release component, while at the other subtype SynII deficiency suppresses the synchronous release component. These results reveal a new SynII function in the regulation of the time course of GABA release and demonstrate that this function is dependent on the interneuron subtype.


Assuntos
Hipocampo/citologia , Interneurônios/fisiologia , Sinapsinas/metabolismo , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Animais Recém-Nascidos , Bloqueadores dos Canais de Cálcio/farmacologia , Estimulação Elétrica , Feminino , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Parvalbuminas/genética , Parvalbuminas/metabolismo , Sinapses , Sinapsinas/genética , Transmissão Sináptica/efeitos dos fármacos , ômega-Agatoxina IVA/farmacologia , ômega-Conotoxina GVIA/farmacologia
7.
J Neurophysiol ; 116(6): 2550-2563, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27605536

RESUMO

The coding of sound level by ensembles of neurons improves the accuracy with which listeners identify how loud a sound is. In the auditory system, the rate at which neurons fire in response to changes in sound level is shaped by local networks. Voltage-gated conductances alter local output by regulating neuronal firing, but their role in modulating responses to sound level is unclear. We tested the effects of L-type calcium channels (CaL: CaV1.1-1.4) on sound-level coding in the central nucleus of the inferior colliculus (ICC) in the auditory midbrain. We characterized the contribution of CaL to the total calcium current in brain slices and then examined its effects on rate-level functions (RLFs) in vivo using single-unit recordings in awake mice. CaL is a high-threshold current and comprises ∼50% of the total calcium current in ICC neurons. In vivo, CaL activates at sound levels that evoke high firing rates. In RLFs that increase monotonically with sound level, CaL boosts spike rates at high sound levels and increases the maximum firing rate achieved. In different populations of RLFs that change nonmonotonically with sound level, CaL either suppresses or enhances firing at sound levels that evoke maximum firing. CaL multiplies the gain of monotonic RLFs with dynamic range and divides the gain of nonmonotonic RLFs with the width of the RLF. These results suggest that a single broad class of calcium channels activates enhancing and suppressing local circuits to regulate the sensitivity of neuronal populations to sound level.


Assuntos
Potenciais de Ação/fisiologia , Canais de Cálcio Tipo L/metabolismo , Colículos Inferiores/citologia , Neurônios/fisiologia , Som , 4-Aminopiridina/análogos & derivados , 4-Aminopiridina/farmacologia , Estimulação Acústica , Potenciais de Ação/efeitos dos fármacos , Amifampridina , Animais , Fenômenos Biofísicos/efeitos dos fármacos , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos CBA , Nimodipina/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Quinoxalinas/farmacologia , Vigília , ômega-Conotoxina GVIA/farmacologia
8.
J Neurosci ; 36(37): 9722-38, 2016 09 14.
Artigo em Inglês | MEDLINE | ID: mdl-27629721

RESUMO

UNLABELLED: Spinal plasticity, a key process mediating neuropathic pain development, requires ubiquitination-dependent protein turnover. Presynaptic active zone proteins have a crucial role in regulating vesicle exocytosis, which is essential for synaptic plasticity. Nevertheless, the mechanism for ubiquitination-regulated turnover of presynaptic active zone proteins in the progression of spinal plasticity-associated neuropathic pain remains unclear. Here, after research involving Sprague Dawley rats, we reported that spinal nerve ligation (SNL), in addition to causing allodynia, enhances the Rab3-interactive molecule-1α (RIM1α), a major active zone protein presumed to regulate neural plasticity, specifically in the synaptic plasma membranes (SPMs) of the ipsilateral dorsal horn. Spinal RIM1α-associated allodynia was mediated by Fbxo3, which abates Fbxl2-dependent RIM1α ubiquitination. Subsequently, following deubiquitination, enhanced RIM1α directly binds to CaV2.2, resulting in increased CaV2.2 expression in the SPMs of the dorsal horn. While exhibiting no effect on Fbxo3/Fbxl2 signaling, the focal knockdown of spinal RIM1α expression reversed the SNL-induced allodynia and increased spontaneous EPSC (sEPSC) frequency by suppressing RIM1α-facilitated CaV2.2 expression in the dorsal horn. Intrathecal applications of BC-1215 (a Fbxo3 activity inhibitor), Fbxl2 mRNA-targeting small-interfering RNA, and ω-conotoxin GVIA (a CaV2.2 blocker) attenuated RIM1α upregulation, enhanced RIM1α expression, and exhibited no effect on RIM1α expression, respectively. These results confirm the prediction that spinal presynaptic Fbxo3-dependent Fbxl2 ubiquitination promotes the subsequent RIM1α/CaV2.2 cascade in SNL-induced neuropathic pain. Our findings identify a role of the presynaptic active zone protein in pain-associated plasticity. That is, RIM1α-facilitated CaV2.2 expression plays a role in the downstream signaling of Fbxo3-dependent Fbxl2 ubiquitination/degradation to promote spinal plasticity underlying the progression of nociceptive hypersensitivity following neuropathic injury. SIGNIFICANCE STATEMENT: Ubiquitination is a well known process required for protein degradation. Studies investigating pain pathology have demonstrated that ubiquitination contributes to chronic pain by regulating the turnover of synaptic proteins. Here, we found that the spinal presynaptic active zone protein Rab3-interactive molecule-1α (RIM1α) participates in neuropathic pain development by binding to and upregulating the expression of CaV2.2. In addition, Fbxo3 modifies this pathway by inhibiting Fbxl2-mediated RIM1α ubiquitination, suggesting that presynaptic protein ubiquitination makes a crucial contribution to the development of neuropathic pain. Research in this area, now in its infancy, could potentially provide a novel therapeutic strategy for pain relief.


Assuntos
Canais de Cálcio Tipo N/metabolismo , Proteínas F-Box/metabolismo , Hiperalgesia/metabolismo , Proteínas rab3 de Ligação ao GTP/metabolismo , Potenciais de Ação/fisiologia , Animais , Benzilaminas/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Modelos Animais de Doenças , Proteínas F-Box/antagonistas & inibidores , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperalgesia/etiologia , Masculino , Neuralgia/complicações , Neurônios/fisiologia , Medição da Dor , Piridinas/farmacologia , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Corno Dorsal da Medula Espinal/efeitos dos fármacos , Corno Dorsal da Medula Espinal/metabolismo , Nervos Espinhais/citologia , Nervos Espinhais/lesões , Nervos Espinhais/metabolismo , Ubiquitinação/efeitos dos fármacos , Ubiquitinação/fisiologia , ômega-Conotoxina GVIA/farmacologia
9.
J Neurophysiol ; 116(2): 456-65, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27146987

RESUMO

BK channels are large-conductance calcium- and voltage-activated potassium channels with diverse properties. Knockout of the accessory BK ß4-subunit in hippocampus dentate gyrus granule neurons causes BK channels to change properties from slow-gated type II channels to fast-gated type I channels that sharpen the action potential, increase the fast afterhyperpolarization (fAHP) amplitude, and increase spike frequency. Here we studied the calcium channels that contribute to fast-gated BK channel activation and increased excitability of ß4 knockout neurons. By using pharmacological blockers during current-clamp recording, we find that BK channel activation during the fAHP is dependent on ryanodine receptor activation. In contrast, L-type calcium channel blocker (nifedipine) affects the BK channel-dependent repolarization phase of the action potential but has no effect on the fAHP. Reducing BK channel activation during the repolarization phase with nifedipine, or during the fAHP with ryanodine, indicated that it is the BK-mediated increase of the fAHP that confers proexcitatory effects. The proexcitatory role of the fAHP was corroborated using dynamic current clamp. Increase or decrease of the fAHP amplitude during spiking revealed an inverse relationship between fAHP amplitude and interspike interval. Finally, we show that the seizure-prone ryanodine receptor gain-of-function (R2474S) knockin mice have an unaltered repolarization phase but larger fAHP and increased AP frequency compared with their control littermates. In summary, these results indicate that an important role of the ß4-subunit is to reduce ryanodine receptor-BK channel functional coupling during the fAHP component of the action potential, thereby decreasing excitability of dentate gyrus neurons.


Assuntos
Potenciais de Ação/genética , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/deficiência , Neurônios/fisiologia , Canal de Liberação de Cálcio do Receptor de Rianodina/deficiência , Potenciais de Ação/efeitos dos fármacos , Animais , Biofísica , Bloqueadores dos Canais de Cálcio/farmacologia , Giro Denteado/citologia , Estimulação Elétrica , Técnicas In Vitro , Indóis/farmacologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Nifedipino/farmacologia , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Rianodina/farmacologia , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , ômega-Conotoxina GVIA/farmacologia
10.
J Neurophysiol ; 116(2): 686-97, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27193322

RESUMO

Horizontal cells form the first laterally interacting network of inhibitory interneurons in the retina. Dopamine released onto horizontal cells under photic and circadian control modulates horizontal cell function. Using isolated, identified horizontal cells from a connexin-57-iCre × ROSA26-tdTomato transgenic mouse line, we investigated dopaminergic modulation of calcium channel currents (ICa) with whole cell patch-clamp techniques. Dopamine (10 µM) blocked 27% of steady-state ICa, an action blunted to 9% in the presence of the L-type Ca channel blocker verapamil (50 µM). The dopamine type 1 receptor (D1R) agonist SKF38393 (20 µM) inhibited ICa by 24%. The D1R antagonist SCH23390 (20 µM) reduced dopamine and SKF38393 inhibition. Dopamine slowed ICa activation, blocking ICa by 38% early in a voltage step. Enhanced early inhibition of ICa was eliminated by applying voltage prepulses to +120 mV for 100 ms, increasing ICa by 31% and 11% for early and steady-state currents, respectively. Voltage-dependent facilitation of ICa and block of dopamine inhibition after preincubation with a Gßγ-blocking peptide suggested involvement of Gßγ proteins in the D1R-mediated modulation. When the G protein activator guanosine 5'-O-(3-thiotriphosphate) (GTPγS) was added intracellularly, ICa was smaller and showed the same slowed kinetics seen during D1R activation. With GTPγS in the pipette, additional block of ICa by dopamine was only 6%. Strong depolarizing voltage prepulses restored the GTPγS-reduced early ICa amplitude by 36% and steady-state ICa amplitude by 3%. These results suggest that dopaminergic inhibition of ICa via D1Rs is primarily mediated through the action of Gßγ proteins in horizontal cells.


Assuntos
Canais de Cálcio/fisiologia , Potenciais da Membrana/fisiologia , Receptores de Dopamina D1/metabolismo , Células Horizontais da Retina/fisiologia , 2,3,4,5-Tetra-Hidro-7,8-Di-Hidroxi-1-Fenil-1H-3-Benzazepina/farmacologia , Animais , Fenômenos Biofísicos/efeitos dos fármacos , Fenômenos Biofísicos/genética , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Conexinas/genética , Conexinas/metabolismo , Dopamina/farmacologia , Agonistas de Dopamina/farmacologia , Antagonistas de Dopamina/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Lectinas de Plantas/genética , Lectinas de Plantas/metabolismo , RNA não Traduzido/genética , RNA não Traduzido/metabolismo , Retina/citologia , Células Horizontais da Retina/efeitos dos fármacos , Espiperona/farmacologia , ômega-Conotoxina GVIA/farmacologia
11.
J Neurosci ; 35(15): 5891-903, 2015 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-25878262

RESUMO

As auxiliary subunits of voltage-gated Ca(2+) channels, the α2δ proteins modulate membrane trafficking of the channels and their localization to specific presynaptic sites. Following nerve injury, upregulation of the α2δ-1 subunit in sensory dorsal root ganglion neurons contributes to the generation of chronic pain states; however, very little is known about the underlying molecular mechanisms. Here we show that the increased expression of α2δ-1 in rat sensory neurons leads to prolonged Ca(2+) responses evoked by membrane depolarization. This mechanism is coupled to CaV2.2 channel-mediated responses, as it is blocked by a ω-conotoxin GVIA application. Once initiated, the prolonged Ca(2+) transients are not dependent on extracellular Ca(2+) and do not require Ca(2+) release from the endoplasmic reticulum. The selective inhibition of mitochondrial Ca(2+) uptake demonstrates that α2δ-1-mediated prolonged Ca(2+) signals are buffered by mitochondria, preferentially activated by Ca(2+) influx through CaV2.2 channels. Thus, by controlling channel abundance at the plasma membrane, the α2δ-1 subunit has a major impact on the organization of depolarization-induced intracellular Ca(2+) signaling in dorsal root ganglion neurons.


Assuntos
Canais de Cálcio Tipo N/genética , Sinalização do Cálcio/fisiologia , Células Receptoras Sensoriais/metabolismo , Regulação para Cima/fisiologia , Animais , Animais Recém-Nascidos , Antimicina A/análogos & derivados , Antimicina A/farmacologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Feminino , Gânglios Espinais/citologia , Indóis/farmacologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Nifedipino/farmacologia , Cloreto de Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Regulação para Cima/efeitos dos fármacos , ômega-Conotoxina GVIA/farmacologia
12.
Mar Drugs ; 13(4): 2030-45, 2015 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-25871286

RESUMO

A set of fluorophenoxyanilides, designed to be simplified analogues of previously reported ω-conotoxin GVIA mimetics, were prepared and tested for N-type calcium channel inhibition in a SH-SY5Y neuroblastoma FLIPR assay. N-type or Cav2.2 channel is a validated target for the treatment of refractory chronic pain. Despite being significantly less complex than the originally designed mimetics, up to a seven-fold improvement in activity was observed.


Assuntos
Analgésicos não Entorpecentes/farmacologia , Anilidas/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo N/metabolismo , Desenho de Fármacos , Proteínas do Tecido Nervoso/antagonistas & inibidores , Neurônios/efeitos dos fármacos , Analgésicos não Entorpecentes/síntese química , Analgésicos não Entorpecentes/química , Analgésicos não Entorpecentes/metabolismo , Anilidas/síntese química , Anilidas/química , Anilidas/metabolismo , Ligação Competitiva , Bloqueadores dos Canais de Cálcio/síntese química , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/metabolismo , Canais de Cálcio Tipo N/química , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Fluorbenzenos/síntese química , Fluorbenzenos/química , Fluorbenzenos/metabolismo , Fluorbenzenos/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Estrutura Molecular , Terapia de Alvo Molecular , Proteínas do Tecido Nervoso/metabolismo , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Neurônios/metabolismo , Neurotoxinas/química , Dor Intratável/tratamento farmacológico , Dor Intratável/metabolismo , Relação Estrutura-Atividade , ômega-Conotoxina GVIA/química , ômega-Conotoxina GVIA/metabolismo , ômega-Conotoxina GVIA/farmacologia
13.
Eur J Pain ; 19(5): 639-48, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25158907

RESUMO

BACKGROUND: Overexpression of the voltage-gated calcium channel (VGCC) alpha-2-delta1 subunit protein (Cav α2 δ1 ) has been shown to cause pain states. However, whether VGCC are involved in pain states driven by abnormal Cav α2 δ1 expression is not known. METHODS: Intrathecal injection of N-, P/Q- and L-type VGCC blockers were tested in two models: a transgenic neuronal Cav α2 δ1 overexpression (TG) model with behavioural hypersensitivity and a spinal nerve ligation (SNL) model with Cav α2 δ1 overexpression in sensory pathways and neuropathy pain states. RESULTS: The nociceptive response to mechanical stimuli was significantly attenuated in both models with ω-conotoxin GVIA (an N-type VGCC blocker) and nifedipine (an L-type VGCC blocker), in which ω-conotoxin GVIA appeared more potent than nifedipine. Treatments with ω-agatoxin IVA (P-VGCC blocker), but not ω-conotoxin MVIIC (Q-VGCC blocker) had similar potency in the TG model as the N-type VGCC blocker, while both ω-agatoxin IVA and ω-conotoxin MVIIC had minimal effects in the SNL model compared with controls. CONCLUSION: These findings suggest that, at the spinal level, N- and L-type VGCC are likely involved in behavioural hypersensitivity states driven by Cav α2 δ1 overexpression. Q-type VGCC has minimal effects in both models. The anti-nociceptive effects of P-type VGCC blocker in the Cav α2 δ1 TG mice, but minimally at the SNL model with presynaptic Cav α2 δ1 up-regulation, suggest that its potential action site(s) is at the post-synaptic and/or supraspinal level. These findings support that N-, L- and P/Q-type VGCC have differential contributions to behavioural hypersensitivity modulated by Cav α2 δ1 dysregulation at the spinal cord level.


Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Nociceptividade/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Canais de Cálcio/genética , Canais de Cálcio Tipo L/efeitos dos fármacos , Canais de Cálcio Tipo N/efeitos dos fármacos , Vias Eferentes/metabolismo , Hiperalgesia/psicologia , Injeções Espinhais , Ligadura , Masculino , Camundongos , Neuralgia/patologia , Neuralgia/fisiopatologia , Nifedipino/farmacologia , Medição da Dor/efeitos dos fármacos , Nervos Espinhais/lesões , ômega-Conotoxina GVIA/farmacologia
14.
Toxicon ; 91: 135-44, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25301479

RESUMO

Conus geographus is the most dangerous cone snail species known, with reported human fatality rates as high as 65%. Crude venom gland extracts have been used to determine animal LD50 and to aid the isolation of several potent paralytic toxins. However, not only is the composition of injected venoms known to differ significantly from that in dissected venom glands, but also to vary according to predatory or defensive stimuli. Therefore, to study the venom that is directly relevant to human envenomation, the defense-evoked venom of several specimens of C. geographus was collected and analyzed by standard LC-MS methods. The molecular composition of individual defense-evoked venom showed significant intraspecific variations, but a core of paralytic conotoxins including α-GI, α-GII, µ-GIIIA, ω-GVIA and ω-GVIIA was always present in large amounts, consistent with the symptomology and high fatality rate in humans. Differences between injected and dissected venoms obtained from the same specimen were also evident. Interestingly, an apparent linear correlation between the dry weight/volume of injected venom and the size of the shell allowed extrapolation to a human lethal dose (0.038-0.029 mg/kg) from an historic fatal case of C. geographus envenomation, which may help in the management of future victims.


Assuntos
Caramujo Conus/química , ômega-Conotoxina GVIA/toxicidade , Animais , Cromatografia Líquida , Caramujo Conus/classificação , Humanos , Proteômica , Especificidade da Espécie , Espectrometria de Massas por Ionização por Electrospray , ômega-Conotoxina GVIA/química
15.
Eur J Neurosci ; 40(5): 2755-65, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24943127

RESUMO

The melanocortin 4 receptor (MC4R) is a G protein-coupled receptor involved in food intake and energy expenditure regulation. MC4R activation modifies neuronal activity but the molecular mechanisms by which this regulation occurs remain unclear. Here, we tested the hypothesis that MC4R activation regulates the activity of voltage-gated calcium channels and, as a consequence, synaptic activity. We also tested whether the proposed effect occurs in the amygdala, a brain area known to mediate the anorexigenic actions of MC4R signaling. Using the patch-clamp technique, we found that the activation of MC4R with its agonist melanotan II specifically inhibited 34.5 ± 1.5% of N-type calcium currents in transiently transfected HEK293 cells. This inhibition was concentration-dependent, voltage-independent and occluded by the Gαs pathway inhibitor cholera toxin. Moreover, we found that melanotan II specifically inhibited 25.9 ± 2.0% of native N-type calcium currents and 55.4 ± 14.4% of evoked inhibitory postsynaptic currents in mouse cultured amygdala neurons. In vivo, we found that the MC4R agonist RO27-3225 increased the marker of cellular activity c-Fos in several components of the amygdala, whereas the N-type channel blocker ω conotoxin GVIA increased c-Fos expression exclusively in the central subdivision of the amygdala. Thus, MC4R specifically inhibited the presynaptic N-type channel subtype, and this inhibition may be important for the effects of melanocortin in the central subdivision of the amygdala.


Assuntos
Tonsila do Cerebelo/fisiologia , Canais de Cálcio Tipo N/metabolismo , Terminações Pré-Sinápticas/fisiologia , Receptor Tipo 4 de Melanocortina/metabolismo , Tonsila do Cerebelo/efeitos dos fármacos , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Fármacos do Sistema Nervoso Central/farmacologia , Toxina da Cólera/farmacologia , Células HEK293 , Humanos , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Masculino , Camundongos , Peptídeos/farmacologia , Peptídeos Cíclicos/metabolismo , Terminações Pré-Sinápticas/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptor Tipo 4 de Melanocortina/agonistas , alfa-MSH/análogos & derivados , alfa-MSH/metabolismo , ômega-Conotoxina GVIA/farmacologia
16.
J Neurosci ; 34(23): 7958-63, 2014 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-24899717

RESUMO

CB1 cannabinoid receptors (CB1) are located at axon terminals and effectively control synaptic communication and thereby circuit operation widespread in the CNS. Although it is partially uncovered how CB1 activation leads to the reduction of synaptic excitation, the mechanisms of the decrease of GABA release upon activation of these cannabinoid receptors remain elusive. To determine the mechanisms underlying the suppression of synaptic transmission by CB1 at GABAergic synapses, we recorded unitary IPSCs (uIPSCs) at cholecystokinin-expressing interneuron-pyramidal cell connections and imaged presynaptic [Ca(2+)] transients in mouse hippocampal slices. Our results reveal a power function with an exponent of 2.2 between the amplitude of uIPSCs and intrabouton [Ca(2+)]. Altering CB1 function by either increasing endocannabinoid production or removing its tonic activity allowed us to demonstrate that CB1 controls GABA release by inhibiting Ca(2+) entry into presynaptic axon terminals via N-type (Cav2.2) Ca(2+) channels. These results provide evidence for modulation of intrabouton Ca(2+) influx into GABAergic axon terminals by CB1, leading to the effective suppression of synaptic inhibition.


Assuntos
Cálcio/metabolismo , Terminações Pré-Sinápticas/metabolismo , Receptor CB1 de Canabinoide/metabolismo , Sinapses/fisiologia , Ácido gama-Aminobutírico/metabolismo , Animais , Animais Recém-Nascidos , Bloqueadores dos Canais de Cálcio/farmacologia , Colecistocinina/genética , Colecistocinina/farmacologia , Feminino , Hipocampo/citologia , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/fisiologia , Interneurônios/efeitos dos fármacos , Interneurônios/fisiologia , Proteínas Luminescentes/genética , Masculino , Camundongos , Camundongos Transgênicos , Piperidinas/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , Pirazóis/farmacologia , Sinapses/efeitos dos fármacos , ômega-Conotoxina GVIA/farmacologia
17.
Neuron ; 82(1): 181-94, 2014 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-24698275

RESUMO

Voltage-dependent Ca(2+) channels (CaVs) represent the principal source of Ca(2+) ions that trigger evoked neurotransmitter release from presynaptic boutons. Ca(2+) influx is mediated mainly via CaV2.1 (P/Q-type) and CaV2.2 (N-type) channels, which differ in their properties. Their relative contribution to synaptic transmission changes during development and tunes neurotransmission during synaptic plasticity. The mechanism of differential recruitment of CaV2.1 and CaV2.2 to release sites is largely unknown. Here, we show that the presynaptic scaffolding protein Bassoon localizes specifically CaV2.1 to active zones via molecular interaction with the RIM-binding proteins (RBPs). A genetic deletion of Bassoon or an acute interference with Bassoon-RBP interaction reduces synaptic abundance of CaV2.1, weakens P/Q-type Ca(2+) current-driven synaptic transmission, and results in higher relative contribution of neurotransmission dependent on CaV2.2. These data establish Bassoon as a major regulator of the molecular composition of the presynaptic neurotransmitter release sites.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Canais de Cálcio Tipo N/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Terminações Pré-Sinápticas/metabolismo , Animais , Células COS , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular Transformada , Chlorocebus aethiops , Exocitose/efeitos dos fármacos , Exocitose/fisiologia , Técnicas In Vitro , Camundongos Transgênicos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Ligação Proteica/fisiologia , Transporte Proteico/efeitos dos fármacos , Transporte Proteico/genética , Vesículas Sinápticas/efeitos dos fármacos , Fatores de Tempo , ômega-Conotoxina GVIA/farmacologia , Domínios de Homologia de src/fisiologia
18.
Mol Pain ; 9: 58, 2013 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-24228737

RESUMO

Voltage gated calcium channels (VGCCs) are well known for its importance in synaptic transmission in the peripheral and central nervous system. However, the role of different VGCCs in the anterior cingulate cortex (ACC) has not been studied. Here, we use a multi-electrode array recording system (MED64) to study the contribution of different types of calcium channels in glutamatergic excitatory synaptic transmission in the ACC. We found that only the N-type calcium channel blocker ω-conotoxin-GVIA (ω-Ctx-GVIA) produced a great inhibition of basal synaptic transmission, especially in the superficial layer. Other calcium channel blockers that act on L-, P/Q-, R-, and T-type had no effect. We also tested the effects of several neuromodulators with or without ω-Ctx-GVIA. We found that N-type VGCC contributed partially to (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid- and (R)-Baclofen-induced synaptic inhibition. By contrast, the inhibitory effects of 2-Chloroadenosine and carbamoylcholine chloride did not differ with or without ω-Ctx-GVIA, indicating that they may act through other mechanisms. Our results provide strong evidence that N-type VGCCs mediate fast synaptic transmission in the ACC.


Assuntos
Canais de Cálcio Tipo N/metabolismo , Giro do Cíngulo/metabolismo , Transmissão Sináptica/efeitos dos fármacos , 2-Cloroadenosina/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Carbacol/farmacologia , Giro do Cíngulo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , ômega-Conotoxina GVIA/farmacologia
19.
Neuroscience ; 253: 316-29, 2013 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-24025868

RESUMO

Presynaptic functions of the mammalian central neurons are regulated by a network of protein interactions. Synaptic vesicle recycling in and neurotransmitter release from the presynaptic nerve terminals are altered when a glutamate-deleting mutation is present in the torsinA protein (ΔE-torsinA). This mutation is linked with a hereditary form of the movement disorder dystonia known as DYT1 dystonia. Although torsinA expression is prevalent throughout the central nervous system, its subcellular localization - in particular with respect to presynaptic nerve terminals - remains unclear. This information would be useful in narrowing down possible models for how wild-type torsinA affects presynaptic function, as well as the nature of the presynaptic dysfunction that arises in the context of ΔE-torsinA mutation. Here we report on an analysis of the presynaptic localization of torsinA in cultured neurons obtained from a knock-in mouse model of DYT1 dystonia. Primary cultures of neurons were established from heterozygous and homozygous ΔE-torsinA knock-in mice, as well as from their wild-type littermates. Neurons were obtained from the striatum, cerebral cortex and hippocampus of these mice, and were subjected to immunocytochemistry. This analysis revealed the expression of both proteins in the somata and dendrites. However, neither the nerve terminals nor axonal shafts were immunoreactive. These results were confirmed by fluorogram-based quantitation. Our findings indicate that neither the wild-type nor the ΔE-torsinA mutant protein is present at substantial levels in the presynaptic structures of cultured neurons. Thus, the effects of torsinA, in wild-type and mutant forms, appear to influence presynaptic function indirectly, without residing in presynaptic structures.


Assuntos
Hipocampo/citologia , Chaperonas Moleculares/metabolismo , Terminações Nervosas/metabolismo , Neurônios/citologia , Análise de Variância , Animais , Cloreto de Cádmio/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Imunofluorescência , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/efeitos dos fármacos , Potássio/farmacologia , ômega-Agatoxina IVA/farmacologia , ômega-Conotoxina GVIA/farmacologia
20.
Neuroscience ; 253: 330-40, 2013 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-24012836

RESUMO

Neurotransmitter release probability is related by high power to the local concentration of calcium in presynaptic terminals, which in turn is controlled by voltage-gated calcium channels. P/Q- and N-type channels trigger synaptic transmission in the majority of neurons of the central nervous system. However, whether and under which conditions both channel types act cooperatively or independently is still insufficiently understood. Previous studies suggested either a dominance of N- or P/Q-type channels, or a synergistic action of both channels, depending on the experimental paradigms. Thus, to provide insight into the properties of neurotransmitter release in cultured mouse hippocampal neurons, we used quantitative analysis of FM dye release from presynaptic boutons induced by high potassium membrane depolarization. Increasing extracellular potassium concentrations revealed a sigmoid dependence of FM dye release to the stimulation strength. Individual and combined application of the P/Q- and N-type channel-specific blockers ω-agatoxin-IVA and ω-conotoxin-GVIA, respectively, allowed us to specifically isolate the contribution of both channel types to release triggered with 40 mM KCl. Analysis of the release kinetics and the fractional release amplitude demonstrate that, whereas in only 15% of the synapses release depended exclusively on P/Q-type channels, the majority of synapses (85%) contained both N- and P/Q-type channels. Nevertheless, the kinetics of FM dye release in synapses containing both channel types was determined by the P/Q-type channels. Together, our data suggest a more direct coupling of P/Q-type channels to synaptic release compared to N-type channels, which may explain the high prevalence of neurological P/Q-type channelopathies.


Assuntos
Canais de Cálcio Tipo N/metabolismo , Corantes Fluorescentes/farmacocinética , Hipocampo/citologia , Neurônios/citologia , Terminações Pré-Sinápticas/metabolismo , Compostos de Piridínio/farmacocinética , Compostos de Amônio Quaternário/farmacocinética , Animais , Cloreto de Cádmio/farmacologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Células Cultivadas , Relação Dose-Resposta a Droga , Embrião de Mamíferos , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Cloreto de Potássio/farmacologia , Terminações Pré-Sinápticas/efeitos dos fármacos , ômega-Agatoxina IVA/farmacologia , ômega-Conotoxina GVIA/farmacologia
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